Protocol to probe how promoters decode TF dynamics in Saccharomyces cerevisiae by combining optogenetic control with microscopy

Abstract

Summary: Gene promoters filter transcription factor (TF) localization dynamics and changes in the DNA binding affinity of TFs. Here, we present a protocol to probe how promoters decode TF dynamics in Saccharomyces cerevisiae by combining optogenetic control with microscopy. We describe steps for preparing and characterizing a light delivery platform and light-controlled TF mutants. We then detail procedures for subjecting the TFs to light doses that generate defined patterns of localization while measuring fluorescent reporter gene activation via live-cell microscopy.For complete details on the use and execution of this protocol, please refer to Sweeney and McClean.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell Biology
• Systems biology
• Biotechnology and bioengineering